The role of nonmuscle myosin in vertebrate cells was explored by expression of a cDNA for the nonmuscle myosin heavy chain in tissue cultured cells. Nonmuscle myosin II heavy chain isoforms bear some sequence homology to sarcomeric myosin heavy chains, and may be expected to have structural and functional homology as well. Nonmuscle myosin appears to function in contractile events within the cell. Evidence from non-vertebrate eukaryotic systems, including Acanthamoeba, Dictyostelium and Drosophila, implicates nonmuscle myosin in cellular functions such as cytokinesis, capping, cell shape, phagocytosis and morphogenetic movements. In this study, the role of nonmuscle myosin in vertebrates was explored by attempting an antisense knockout of the endogenous protein in skeletal muscle cells. The mouse skeletal muscle satellite cell line, C2C12, was transfected with vectors containing fragments of chicken intestinal epithelial nonmuscle myosin cDNA. The fragments were under the control of the inducible mouse mammary tumor virus promoter. Changes in morphology and differentiation were observed. PCR was used to demonstrate incorporation of the cDNA into the cells' genomic DNA and expression of the inserts into mRNA. Under high serum growth conditions, little effect was seen on growth rate or total protein composition, but a small fraction of the cells underwent a morphological change. They appeared as flattened, rounded discoids with many micronuclei or coalesced nuclei. In low serum, differentiation conditions, changes were seen in total cell protein composition and in the rate of myoblast fusion to form myotubes.